Chapter 19_post_Part 2_

Genetics: A Conceptual Approach

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Chapter 19- Part II Molecular Genetic Analysis and Biotechnology Dr. Ed Otto George Mason University
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Molecular Techniques Used to Analyze DNA Sequences - RFLPs - DNA Sequencing - DNA Fingerprinting
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RFLPs Restriction fragment length polymorphisms (RFLPs) are variations (polymorphisms) in the patterns of fragments produced when DNA molecules are cut with the same restriction enzyme These differences are inherited and can be used in mapping, similar to the way allelic differences are used to map genes that produce phenotypes
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RFLPs Example: Huntington disease Autosomal dominant disorder The disorder causes death (dominant lethal), but its late onset does not preclude affected individuals from passing the trait to offspring The disease-causing form of the Huntington gene ( H allele) is closely linked to a RFLP: digestion with restriction enzyme Hae III produces two bands Digestion of the wild type allele ( h ) produces three bands
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RFLPs Hae III Hae III Hae III Closely linked to wild-type Huntington allele ( h ) Closely linked to mutant Huntington allele ( H )
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DNA Sequencing DNA sequencing is a powerful molecular method for determining the sequence of bases in DNA In 1970’s Fred Sanger invented the dideoxy- sequencing method (a.k.a. enzymatic method), which quickly became the method of choice for sequencing any purified DNA fragment
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This method relies on the use of special nucleotides for DNA synthesis: dideoxyribonucleoside triphosphates (ddNTPs) ddNTPs are similar to dNTPs, except that they lack a 3’-OH In the course of DNA synthesis, when a ddNTP is incorporated into a growing DNA strand, no more nucleotides can be added because there is no 3’-OH group available to form a phosphodiester bond with the incoming nucleotide DNA Sequencing
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Step 1 : Amplify copies of the DNA molecule to be sequenced (sequencing template) by cloning or by PCR Step 2 : Split the sequencing template into four tubes and set up reactions with the following components: DNA template Radiolabelled primer that is complementary to one end of the template All four dNTPs One ddNTP (low concentration) DNA polymerase ddATP ddGTP ddCTP ddTTP Tube A Tube G Tube C Tube T DNA Sequencing
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Example: what’s going on in Tube A - The primer pairs with its complementary sequence at one end of the template - DNA polymerase elongates the primer - Whenever DNA polymerase encounters a T on the template strand, it incorporates at random either dATP or ddATP into the growing strand - Because there is more dATP than ddATP in the reaction mixture, dATP is incorporated more often - Occasionally ddATP is incorporated into the strand, and synthesis terminates - The incorporation of ddATP occurs randomly at different positions in different elongated strands, producing a set of DNA strands of different lengths Step 3 : Incubate the tubes to allow DNA synthesis DNA Sequencing
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This note was uploaded on 05/06/2010 for the course BIO 311 taught by Professor Otto during the Spring '10 term at George Mason.

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Chapter 19_post_Part 2_ - Chapter 19 Part II Molecular...

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